3.8.7 UV-visible spectroscopy

General principles

Absorption of incident radiation by
bonding/non-bonding electrons represents a high energy (~100 kCal/
mole) transition. This corresponds to a high frequency, i.e. low wavelength,
absorption band which is observed at 200 ~ 800 nm in the UV and
visible range of detection. In solution, electronic absorption spectra are
found with broad, generally unresolved bands. These contrast with the vibration
fine structure in the vapour phase and with a series of sharp peaks within a
continuum in non-polar solvents.

For a solution of an absorbing
substance, an absorptivity ratio at a monochromatic wavelength is defined as:
{(incident light, Io)/(transmitted, I)} and this is logarithmically related to
concentration and optical path-length by the Beer Lambert law: Absorbance (A) =
log10(Io/I) = k.c.l., where c mg/ml is the concentration of solute
and 1 cm is the distance travelled between parallel optical faces of a suitable
cell, and k is a proportionality constant. It is frequently convenient to
normalise to a concentration c = 10 mg/ml [i.e. 1%] and l = 1 cm, which is expressed
as the specific absorbance [A1%1cm]. Molar
absorptivity is defined by the coefficient ε =
Mr.(A/cl), and is related to the relative molecular mass,
Mr. This coefficient is computed for each wavelength maximum, and
also at minima if this is of diagnostic value. It may be useful in showing
relationships within an homologous series.

For light source emissivity, the common
radiation source is a deuterium lamp covering the operating range
180~ 350 nm and supplemented by a tungsten filament lamp in the near
UV, through the visible, into the near-IR, i.e. over the range
320~1000 nm. Standardisation of equipment and monochromators is
necessary to ensure the acceptability of data. The wavelength scale is
calibrated with a Holmium perchlorate solution, within a tolerance of ±1
nm below 400 nm and ± 3 nm in the 400~600 range (see British
Pharmacopoeia, 1993). The absorbance may be checked with NPL calibrated neutral
density filters; or should agree within defined corresponding
‘windows’ with absorbances obtained with a potassium dichromate
solution of specified strength, at wavelengths 235, 257, 313 and 350 nm. Stray
light is usually checked with a 1.2% potassium chloride solution, where
the absorbance for 1 cm path length should exceed 2.0 at 200 nm against a water
reference. This solution can be replaced by 1% NaBr or NaI at the more accessible
wavelengths of 215 or 240 nm respectively. Glass optics absorb UV light below
about 300 nm and quartz systems are used to extend the working range down to
200 nm, and even to 185 nm if there are high quality optics and stray light
control. At lower wavelengths, absorption of UV-radiation by air requires the
use of vacuum systems in research instruments. For practical UV-vis
spectrophotometry, the effective working range is 200~800 nm.

Operating conditions

Selection of a suitable solvent is
influenced by the wavelength expected to be studied. Water and the lower
(polar) alcohols, through diethyl ether and dioxan to nonpolar cyclohexane and
light petroleum (‘aromatic free’ in a spectroscopic grade) can be
used above 190 nm, whereas chloroform absorbs below ~245 nm. The table below
provides a list of cut-off wavelengths. Measured absorbances should be less
than 0.4 relative to air using prism monochromators; but higher absorptivity
ratios are favoured with modern instruments.

The choice of cells depends on the
target range. Silica is essential for measurements at UV wavelengths but glass
is acceptable in the visible region; air must be evacuated below
~200 nm. The matched pair required for test solution and solvent
reference path should demonstrate effectively identical absorbance when filled
with the same solvent. The optical faces of the cells should be parallel; the
absorbance of a matched pair of cells containing the same solvent should not
differ by more than 0.005 units. In quantitative work all solutions should be
at the same temperature; conveniently, they are transferred from a waterbath
at, say, 20° and the absorbance measured immediately. Sensitivity of the
solution to laboratory and natural lighting should be established in a pilot
experiment. One test (British Pharmacopoeia, 1993) of Resolution Power is the
discrimination of adjacent maximum (269 nm) and minimum (266 nm) light
absorption of toluene with a ratio not less than 1.5.

Quantitative procedures

UV photometry is a frequently used
assay technique. Provided that proper calibration checks are maintained, the
UV-vis technique is particularly useful for assay of formulations after
extraction or separation of the active substance by suitable chromatography. In
the assay calibration, there may be some deviation from Beer’s Law. This
may be attributable to association in solution or an effect of slit width. The
latter should be large enough to gain a reasonable I-value but remain small
compared with the (half-) bandwidth for the absorption measured. If in doubt,
reduce the slit width slightly and check if the apparent absorbance increases.
UV photometric data can also be of value in determining the kinetics of a
process, or in following a reaction sequence, such as the disappearance of an
absorption peak representing starting material.

Light absorption’ measurements
also provide a semi-quantitative test of identity. This relies on the specific
absorbance (defined above as the A1cm1% value), or sometimes
absorbance at a nominated concentration and path length, either exactly at a
specified wavelength, or at the absorption maximum close to a named wavelength.
If this test is used as the principal assay of a substance in a formulation, it
is advisable to use an authenticated reference substance rather than rely on a
published A11. The absorption spectrum may be sensitive
to control of pH. Chromophores involving an acidic or basic group will be
affected by pH, e.g. the bathochromic shift (to longer wavelength) and
hyperchromic peak (greater intensity) of phenates compared with their parent
phenol. This is a useful test for a phenolic system.

In subtractive spectrophotometry, the
difference between two (or more) spectra measures multicomponent mixtures and
is especially useful in formulated product assays. This should be distinguished
from the use of second derivative spectroscopy, in which there is computer
differentiation of the algebraic function equivalent to the change of slope
(i.e. second differential) of the digitalised spectrum (British Pharmacopoeia,
1993). This display sharpens separation of individual UV bands and thereby
facilitates lower levels of control. In other applications of computer-aided
spectroscopy, modern equipment will provide ‘smoothing’,
deconvolution and regression (least squares) analysis.

General chromophores

Absorption bands are particularly
evident for conjugated π-bond systems. Most single bond
transitions are inaccessible, being derived from higher energy
σ-orbitals, with wavelengths below 185 nm, i.e. in the
‘vacuum ultraviolet’. Many isolated triple bonds also absorb below
185 nm. The CC (~185) and CN
(~190) double bonds exhibit strong
π–π* interactions but
unless there is very good control of stray light, measurement is still
unreliable in this region. At longer wavelengths there are rather weak
n−π*
interactions, such as NO and keto CO in the range 280~300 nm.
Simple benzene compounds show medium intensity multiplets around 254 nm for
non-conjugated derivatives, and shifted to longer wavelengths when substituents
are conjugated to the aromatic system. In the table at the end of this section
there are examples of commonly encountered chromophores, including conjugated
alkene, carbonyl and aromatic systems which exhibit bathochromic (longer
wavelength) and hyperchromic (enhanced absorptivity) changes. For very
extensive catalogues of individual UV spectra, refer to DMS (1960–1971)
(1160 substances), Hirayama (1967) (8500 selected values) and Sadtler (1979)
(2000 spectra for 1600 compounds). Older spectra of specifically aromatic
compounds were collated by Friedel and Orchin (1951). Schemes such as
Woodward’s ‘Rules’ (1941, 1942), as further modified by L.
and M. Fieser, for conjugated polyenes and en-ones, have considerable
predictive power. (For a fuller discussion, see Scott (1964).)

In this table, approximate wavelengths
(nm) are specified below which the solvent absorbance may be unacceptable. For
quantitative work, the cut-off may be set at a wavelength
(L0) where the absorbance for 10 mm pathlength of the solvent
exceeds 0.05 absorbance unit (relative to water), i.e. A1 cm >
0.05. For qualitative work, it may still be feasible to work at
significantly lower wavelengths and most analysts accept a cut-off based on the
wavelength (L1) for A1 cm > 1.0. However, if the UV
absorption curve rises steeply, the accessible wavelength range may not be
greatly extended.

L0

L1

L0

L1

Alcohols

Halocarbons (contd)

methanol

240

205

1,2-dichloroethane

250

230

ethanol

240

205

tetrachloroethylene

320

290

n-propanol

250

210

trichloroethylene

>400

2-propanol

240

205

n-butanol

245

215

Miscellaneous

s-butanol

285

260

acetonitrile

200

190

isobutanol

250

200

NN-dimethylformamide

300

270

dimethylsulphoxide

330

285

Esters

nitromethane

>400

380

ethyl acetate

280

260

pyridine

345

325

n-butyl acetate

275

255

water

190

185

Ethers

Alkanes

diethyl ether

255

220

pentane

230

200

p-dioxane

290

220

hexane

225

195

tetrahydrofuran

280

220

heptane

230

200

2-methoxyethanol

270

200

cyclopentane

220

195

2-ethoxyethanol

280

210

cyclohexane

235

200

1,2-dimethoxyethane

300

220

2,2,4-trimethylpentane

[‘isooctane’]

230

210

Ketones

decalin

250

230

acetone

340

330

butan-2-one [MEK]

345

330

Aromatic hydrocarbons

4-methylpentanone [MIBK]

375

335

benzene

295

280

5-methylhexanone [MIAK]

350

330

toluene

315

285

chlorobenzene

310

285

Halcocarbons

1,2-dichlorobenzene

350

295

chloroform

260

240

o-xylene

325

290

dichloromethane

245

230

1,2,4-trichlorobenzene

350

?

UV absorption bands for typical chromophores

For nonconjugated
π-systems, the bands may
be inaccessible for conventional spectrometers. Conjugated alkene, carbonyl and
aromatic systems are at longer wavelengths and more intense.